The present invention relates to isolated polypeptides having 2,6-xcex2-D-fructan hydrolase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides as well as compositions comprising the polypeptides and the use thereof.
2,6-xcex2-D-fructans, such as levans of bacterial origin and phleins of plant origin, are substantially xcex2-2,6-fructose polysaccharides consisting of a variable number of fructose units combined by xcex2-(2xe2x86x926)-glycosidic linkages.
In phlein, xcex2-2,1 branching points also exist depending on the plant origin. Many plants, in particular grasses, store phlein as reserve polysaccharides in stems and leafs. As grasses are ubiquitous plants, phlein is an attractive resource as it is available in almost unlimited amounts.
Levans originate from bacteria. In nutrient limited ecosystems, bacteria have a marked tendency to adhere to surfaces and initiate the formation of a biofilm. A biofilm is a community of microbes, embedded in an organic polymer matrix, adhering to a surface. In natural and industrial ecosystems, especially in nutrient limited systems, biofilms will predominate and cause problems as increased frictional resistance to fluids in water conduits and on ship hulls (fouling), decreased heat transfer from heat exchangers, corrosion of metallic substrates and contamination in the food and biotechnology industry. Biofilms are also a severe problem in medical science and industry causing dental plaque, contaminated endoscopes and contact lenses, prosthetic device colonization and biofilm formation on medical implants.
A biofilm is a collection of microcolonies, typically with water channels in between, and an assortment of cells and extracellular polymers (polysaccharides, glycoproteins, proteins). Bacterial extracellular polysaccharides are composed of homo- and heteropolysaccharides of particularly glucose, fucose, mannose, galactose, fructose, pyruvate, mannuronic acid or glucuronic acid based complexes. The different bonds between the saccharides give rise to a multitude of different polysaccharides including levans, polymannans, dextrans, cellulose, amylopectin, glycogen and alginate.
Bacteria growing in biofilms are more resistant to antibiotics and disinfectants than planktonic cells and the resistance increases with the age of the biofilm. Bacterial biofilm also exhibits increased physical resistance towards desiccation, extreme temperatures or light. As mentioned, biofilm formation causes industrial, environmental and medical problems and the difficulties in cleaning and disinfection of bacterial biofilm with chemicals is a major concern in many industries. Furthermore, the trend towards milder disinfection and cleaning compositions may increase the insufficient cleaning of surfaces covered with biofilm.
Levansases are known e.g. from Fuchs et al. [Fuchs A, DeBruijn J M and Niedeveld C J; Antonie van Leeuwenhoek, 1985 52 (3) 333-343].
Levanases from bacteria have been reported such as from Arthrobacter species strain 7 by Zelikson R. and Hestrin S., Biochemical Journal (1961), 79 page 71-79, from S. salivarius by Takahishi N, Mizuno F, Takamori K (1983) Infection and Immunicity vol 42, pp 231-236 and from A. viscosus by Igarashi T., Takahashi M., Yamamoto A., Etoh Y., Takamori K., (1987), Infection and immunity, Vol 55, pp. 3001-3005.
A levanase from Bacillus sp. No. L7 was reported by Miasnikov A. N. (Characterization of a novel endo levanase and its gene from bacillus sp. L7; FEMS Microbiology Letters, 1997, vol. 154, pp. 23-28).
An application of a Bacillus sp. No. 7 endo-levanase is described in WO 99/31020.
A levanase from Bacillus polymyxa was reported by Bezzate et al. in J. Bacteriol. (1994), 176(8), 2177-83.
U.S. Pat. No. 3,773,623 describes a composition for treating slime in industrial waters with dead cells of the yeast Rhodotorula sp. having levan hydrolase activity
WO 97/01669 describes a method to remove water binders such as slime on a press felt by spraying enzymes onto or into the press felt.
EP 129315 A1 describes a separately packed two component biocidal system.
A process for the manufacture of fructose is described in JP 52136929 A and JP 52136928. A.
U.S. Pat. No. 4,927,757 describes the manufacture of fructose by use of a fructosyl transferase and a levanase.
KR 9301882 B describes the manufacture of a fructo-oligosaccharide preparation by treating a sucrose containing medium with a levanase.
The present invention relates to an isolated polypeptide having 2,6-xcex2-D-fructan hydrolases activity, selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 55.3% identity with amino acids 32 to 923 for the mature polypeptide of SEQ ID NO:1 or 25 to 1277 for the mature polypeptide of SEQ ID No. 3 or 29 to 943 for the mature polypeptide of SEQ ID No. 5;
(b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with (i) nucleotides 94 to 2769 of SEQ ID NO:2 or 73 to 3824 of SEQ ID No:4 or 85 to 2832 of SEQ ID No:6, (ii) the cDNA sequence comprised in nucleotides 94 to 2769 of SEQ ID NO:2 or 73 to 3824 of SEQ ID No:4 or 85 to 2832 of SEQ ID No:6, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii);
(c) a variant of the polypeptide having an amino acid sequence of SEQ ID NO:1 or SEQ ID No. 3 or SEQ ID No. 5 comprising a substitution, deletion, and/or insertion of one or more amino acids;
(d) an allelic variant of (a) or (b);
(e) a fragment of (a), (b), or (d) that has 2,6-xcex2-D-fructan hydrolases activity; and
(f) a polypeptide having
i) a 2,6-xcex2-D-fructan hydrolase activity optimum in the pH range of 3.5-91.5, measured at 37xc2x0 C.;
ii) a molecular mass greater or equal to about 88 kDa;
iii) a 2,6-xcex2-D-fructan hydrolase activity optimum in the temperature range of 20-70xc2x0 C.
The present invention also relates to isolated nucleic acid sequences encoding the polypeptides and to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides. Also the invention relates to a process for removing a microbial biofilm on a surface comprising contacting said biofilm with an endo-2,6-xcex2-D-fructan hydrolase in an aqueous medium.